Mesenchymal Stem Cells Regulate IL8 and TGFA Expression in a Novel Leucocytes Depleted Platelet-Rich Plasma-Skin Equivalent in a Preliminary in vitro Study of Chronic Wound Healing

Abstract

Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type 2 Diabetes Mellitus, and long-term venous insufficiency. Mesenchymal stem cells (MSCs) ability to modulate the inflammatory response represents the fundamental requisite for their applicability as a treatment of chronic wounds.Methods: This study aimed to develop a novel bioactive platelet-rich plasma (PRP)-leukocytes-depleted scaffold to reproduce typical clinical wound of patients with poor chronic skin perfusion and low leucocytes infiltration. After scratching the wound model to mimic injury three conditions were compared; an untreated condition, a condition treated with recombinant TNF to mimic an inflammatory state and a condition treated with TNF and also with MSCs to evaluate how the latter’s immunomodulatory properties affect the therapeutic outcomes in an inflammatory state. Gene expression of IL8 and TGFA were analysed in biological triplicates of the three conditions. Statistical analysis was done through a paired student t-test and a p <0.05 was considered significant.Results: We set up a skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold, with embedded fibroblasts as dermal equivalent and seeded keratinocytes on it as multi-layered epidermidis. IL8 expression increased upon scratching (p=0.014) and continued to increase up to day 1 (p=0.048). IL8 expression decreased upon administration of TNF (p=0.005) but then increased again. IL8 expression decreased in the untreated condition after day 1 as the natural healing process took place and was lower than in treated conditions in day 8 (p=0.048). TGFA expression decreased upon scratching (p=0.006) and increased again in day 1, more so in the untreated than in the treated conditions (p=0.02). TGFA expression decreased again in day 4 in the study group before increasing sharply (p=0.027) in day 8 to reach pre-scratch levels. Conclusion: This study found that a leukocyte-depleted PRP-based skin equivalent can be useful in the study of treatments of chronic wounds. This study also indicates that MSCs appear to modulate the expression of IL8 by switching from an immunosuppressive phenotype to a pro-inflammatory phenotype. These results indicate that the administration of MSCs could offer a potential therapeutic approach for the treatment of leg ulcers in patients with poor skin perfusion.